Transcript Genotyping of hepatitis A virus (HAV) - a useful tool for outbreak investigations Kathrine Stene-Johansen NIPH.

Genotyping of hepatitis A virus (HAV)
- a useful tool for outbreak
investigations
Kathrine Stene-Johansen
NIPH
Hedmark (2)
Hedmark
Oslo
Marokko
Kristiansand
HAV
Sudan
• Picornavirus family
Israel
Chile 1
• ss RNA viral genome
Brasil
of 7.5 kB Levanger
• The capsid is Chile 2
1995-1999
composed of IVDU-epidemi
3
IVDU-Finland
structural proteins;
MSM-utbrudd 1997/98
VP1, VP2 andLørenskog
VP3
Bergen
• A single serotype
Hellas
Oslo
Østfold, Nølkeby skole (4)
Oslo
Hedmark
Meråker
England
MSM-utbrudd
Tyrkia
USA
0.1
Genome organisation of HAV
Costa-Mattioli M et al. J Gen Virol 84 (2003), 3191-3201
HAV genotype classification
• A genotype is defined as a group of viruses with
> 85% nucleotide identity
• A sub-genotype is defined as a group of viruses
with sequence variability of less than 7.5%
3 human genotypes
– genotype I
– genotype III
– genotype II
• 3 simian genotypes
>90%
< 10%
2 isolates
HAV genotypes
FH3 LY6
AH3
AH2
Genotype
LU38
FH1GBM
FH2
F.G.
AH1
NCACG
HAF-203
Genotype IB
IA
HM-175
L-A-1
1000
1000
PA21 GA76
MBB
P27
1000
1000
Genotype IIIA
NOR21
1000 989 612
Genotype IIA
CF53
Genotype IIB
Genotype IV
SLF88
simian strain
Genotype V
simian strain
AGM27
0.1
CY-145
HAV strains
• High endemic regions
– Endemic HAV populations with closely related
strains and geographic relatedness
• Low endemic regions
– Imported strains from high endemic regions
with diverse origin and relatedness
Genotyping
•
•
•
•
HAV RNA isolation from serum
Reverse transcriptase (rt)
PCR
Sequencing
Molecular epidemiology of HAV
• By sequencing 350-450 bp in the
VP1-2PA region we can distinguish
between outbreak strain
• Within outbreaks HAV sequences are
identical or very closely related so that
epidemiological defined cases are
confirmed or precluded
Notified cases of HAV infected in Norway
1994-2004
700
600
500
Unknown transmission in Norway
400
Outbreak men who have sex with
men
Outbread drug users
O
300
200
100
0
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
Genotyping used for outbreak
investigation in Norway (1995-2004)
• Epidemic among drug users 1995-1998
• Outbreak among homosexual men
1997-98 and 2004
• Imported cases
• Small local outbreaks
• Outbreak among hemophiliacs
Notified cases from January 1995 - July 1998
Genotyping
723 cases associated
with the epidemic
among drug users
1 242 cases
581 drug users
46
54 genotype IA (IVDU-strain I)
65
142 secondary cases
11 genotype IIIA (IVDU-strain II)
19
19 drug users (IVDU- strain I)
519 cases not associated
with the epidemic among
drug users
IVDU= Intravenous drug abusers
49
30 other variants
Nosocomial outbreak
faecal-oral transmission
• A hospitalised alcoholic transmitted the
virus to 14 secondary cases
– 8 nurses
– 4 other patients
– 2 relatives
• Outbreak strain of genotype IA identical to
outbreak strain circulating among drug
users at this time ( IVDU- strain I)
Outbreak among hemophiliacs
•
•
•
•
Parenteral transmission
In 1999 4 hemophiliacs were transmitted with
HAV, where coagulation factor VIII was the
possible source of transmission
2 batches of coagulation factor VIII and
serum from 2 hemophiliacs were analysed
Identical virus were detected among the
hemophiliacs and in the batches with
coagulation factor
This outbreak strain was identical to the
IVDU-strain II (genotype IIIA)
Outbreak among homosexual men
1997-1998
• 26 notified cases of HAV infection among
homosexual men from October 97- March 98
• 18/23 PCR-positive
• Genotyping revealed 2 strains
– 12 MSM-strain I (genotype IA)
– 5 MSM-strain II (genotype IA)
• Outbreak strains distinct from the IVDU-strains
• MSM-strain II was also detected in a local family
outbreak
MSM= men with sexual contact with men
Family Outbreak
Grandfather
Grandmother
IgMOutbreak among homosexual men
Father
+3d/PCR-
Mother
Uncle
+32d/PCR- 0d/PCR+
IgM-
IgM+
18/23 PCR+
Sister
- 67d
+11d
+11d
Sister
+11d
Aunt
Sister
+11d
IgM-/PCR-
Outbreak among homosexual men (2004)
• 79 notified cases of HAV associated with
homosexual men in the period May - November
2004
• HAV RNA detected in 67/79
• Identical sequences of genotype IA were
detected
• The same outbreak strain caused concurrent
outbreaks among homosexual men in Denmark,
Sweden and the Netherlands
Genotype IA
MSM outbreak 1997-98
Bergen
USA
Turkey
Brazil
IVDU outbreak Finland
IVDU epidemic 1995-1998
Chile
Marocco
Genotype IIIA
IVDU epidemic
1997-98
Genotype III B
Indonesia
Kristiansand
Sudan
Israel
Genotype IB
Conclusion
• By molecular epidemiology we have been able to
characterise and distinguish outbreaks of HAV,
as well as studied the dissemination of outbreak
strains in the population.
• These studies have shown that sequencing of
outbreak strains is essential in outbreak
investigation, and that molecular epidemiology is
an excellent tool for the surveillance of HAV.